Human reproductive function is controlled in part by a family of heterodimeric human glycoprotein hormones which have a common alpha subunit, but differ in their hormone-specific beta subunits. The family includes follicle-stimulating hormone (FSH), luteinizing hormone (LH), thyrotropin or thyroid-stimulating hormone (TSH), and human chorionic gonadotropin (CG). In all cases, the alpha subunit is a 92 amino acid glycoprotein with two canonical glycosylation sites at the asparagines located at positions 52 and 78. The beta subunits are also glycoproteins; in addition to the N-linked glycosylation exhibited by the beta chains of all four hormones, human CG contains four mucin-like O-linked oligosaccharides attached to a carboxy-terminal extension unique to this hormone. The relevance of the O-linked glycosylation is not, apparently, related to the secretion and assembly of the hormone (Matzuk, M. M. et al. Proc Natl Acad Sci U.S.A. (1987) 84:6354-6358).
Genomic and cDNA clones have been prepared corresponding to the human alpha chain (Boothby, M. et al. J. Biol Chem (1981) 256:5121-5127; Fiddes, J. C. et al. J Mol App Genet (1981) 1:3-18). The cDNA and/or genomic sequences of the beta subunits have also been prepared. For CG, the beta-encoding DNA is described by Fiddes, J. C. et al. Nature (1980) 286:684-687 and by Policastro, P. et al. J Biol Chem (1983) 258:11492-11499. For luteinizing hormone, such description is by Boorstein, W. R. et al. Nature (1982) 300:419-422; and for TSH by Hayashizaki, Y. et al. FEBS Lett (1985) 188:394-400 and by Whitfield, G. K. et al. in "Frontiers in Thyroidology", (1986) Medeiros-Nato, G. et al. (eds) pages 173-176, Plenum Press, N.Y. These DNA segments have been expressed recombinantly, and biologically active material has been produced.
The genomic sequence encoding FSH beta chain was used to construct a recombinant expression vector containing the complete beta chain coding sequence as described in PCT application WO86/04589, published 14 Aug. 1986. In addition genomic clones for human FSH-beta have been prepared by others (Watkins, P. C. et al. DNA (1987) 6:205-212; Jameson, J. L. et al., Mol Endocrinol (1988) 2:806-815; Jameson, J. L. et al. J Clin Endocrinol Metab (1986) 64:319-327; Glaser, T. et al. Nature (1986) 321:882-887). However, it is not clear that human FSH beta has been engineered to permit recombinant production of the dimeric hormone. (The bovine beta FSH gene has also been obtained as disclosed in Maurer, R. A. et al. DNA (1986) 5:363-369; Kim, K. E. et al. DNA (1988) 7:227-333.)
The invention constructs permit significant recombinant production of this family of hormones, in particular, FSH and LH, and further sets forth a means for regulation of the glycosylation pattern and thereby greater predictability in the formulation of therapeutically useful materials.
While it is now understood that the glycosylation pattern of a particular protein may have considerable relevance to its biological activity, the importance of this pattern has largely been overlooked in characterization of glycoproteins. Emphasis has been placed on the amino acid sequence as if this were the sole component of the glycoprotein. The reasons for this myopia are largely historic, but this almost exclusive focus on the peptide aspect is clearly in error. For example, it is well known in the case of human CG that desialylation causes the hormone to be cleared rapidly via the liver (Morell, A. G. et al. J Biol Chem (1976) 246:1461-1467). It is also known that removal of carbohydrate internal to the sialic acid residues or complete deglycosylation converts human CG into an antagonist which binds more tightly to receptor but shows decreased biological activity in vitro (Channing, C. P. et al. Endocrinol (1978) 103:341-348; Kalyan, N. J. et al. J Biol Chem (1983) 258:67-74; Keutmann, H. T. et al. Biochemistry (1983) 3067-3072; Moyle, W. R. et al. J Biol Chem (1975) 250:9163-9169). Other glycoproteins, such as, for example, tissue plasminogen activator, are also known to be altered in their degree of activity when the glycosylation pattern is changed. Therefore, it appears that in order to regulate the therapeutic function of the glycoprotein hormones, it may be necessary to control both the level and nature of glycosylation.
Human FSH and LH are used therapeutically to regulate various aspects of metabolism pertinent to reproduction in the human female. For example, FSH partially purified from urine is used clinically to stimulate follicular maturation in anovulatory women with anovulatory syndrome or luteal phase deficiency. Luteinizing hormone (LH) and FSH are used in combination to stimulate the development of ovarian follicles for in vitro fertilization. The role of FSH in the reproductive cycle is sufficiently well-known to permit this sort of therapeutic use, but difficulties have been encountered due, in part, to the heterogeneity and impurity of the preparation from native sources. This heterogeneity is due to variations in glycosylation pattern. Similarly, LH is used therapeutically to alleviate conditions associated with impaired functioning of the female reproductive system.